| Research Abstract |
To study the role of Pro residues in the conformation and conformational stability of a protein, nine mutant alpha-subunits of tryptophan synthase from Escherichia coli, in which Ala or Gly was substituted for each of six conserved Pro residues (positions 28, 57, 62, 96, 132 and 207) in 10 microorganisms, were examined by CD (Jasco J500), scanning microcalorimetric (DASM4), and stopped flow (RA-401) measurements. Calorimetric measurements showed that the wild- type and examined mutant proteins, exceptions being the two mutants at position 28 (P28G and P28A), each gave a single peak in the excess heat capacity curve. P28G and P28A gave two peaks, suggesting that the substitutions at Pro-28 affected the denaturation process of the alpha-subunit. The stabilities of the Ala mutants,at positions 57, 62, and 132 were a little lower than that of the wild-type. The stabilities of the Ala mutants at positions 96 and 207 were remarkably decreased. The denaturation enthalpy changes, at the same temperature, of the examined mutants, except for P207A, were similar to in the case of the wild-type, indicating that the differences in denaturation Gibbs energy among mutant proteins are caused by an entropic factor. The present results suggest that the conserved Pro residues of the tryptophan synthase alpha-subunit play roles in the conformational stability and protein folding, among other significant functions.
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